Review ArticleOrigins & Design 17:1

The RNA World: A Critique

Dean Kenyon
Department of Biology
San Francisco State University
1600 Holloway Avenue
San Francisco, CA 94132

Introduction

One of the earliest published suggestions that RNA-catalyzed
RNA replication preceded and gave rise to the first DNA-based
living cells was made by Carl Woese in 1967, in his book The
Genetic Code1. Similar
suggestions were made by Crick and Orgel2, for reasons that are not difficult to grasp.
Prior to the discovery of catalytic RNAs, proteins were considered
by many to be the only organic molecules in living matter that
could function as catalysts. DNA carries the genetic information
required for the synthesis of proteins. The replication and transcription
of DNA require a complex set of enzymes and other proteins. How
then could the first living cells with DNA-based molecular biology
have originated by spontaneous chemical processes on the prebiotic
Earth? Primordial DNA synthesis would have required the presence
of specific enzymes, but how could these enzymes be synthesized
without the genetic information in DNA and without RNA for translating
that information into the amino acid sequence of the protein enzymes?
In other words, proteins are required for DNA synthesis and DNA
is required for protein synthesis.

This classic "chicken-and-egg" problem made it immensely
difficult to conceive of any plausible prebiotic chemical pathway
to the molecular biological system. Certainly no such chemical
pathway had been demonstrated experimentally by the early 1960s.
So the suggestion that RNA molecules might have formed the first
self-replicating chemical systems on the primitive Earth seemed
a natural one, given the unique properties of these substances.

They carry genetic information and (unlike DNA) occur primarily
as single-stranded molecules that can assume a great variety of
tertiary structures, and might therefore be capable of catalysis,
in a manner similar to that of proteins. The problem of which
came first, DNA or proteins, would then be resolved.

Self-replicating RNA-based systems would have arisen first,
and DNA and proteins would have been added later. But in the absence
of any direct demonstration of RNA catalysis, this suggestion
remained only an interesting possibility.

Then, in the early 1980s3,
the discovery of self-splicing, catalytic RNA molecules (in the
ciliated protozoan Tetrahymena thermophila), put molecular
flesh on the speculative bones of the idea of an early evolutionary
stage dominated by RNA. These catalytic RNA molecules have subsequently
been termed "ribozymes." "One can contemplate an
RNA World," wrote Walter Gilbert in 1986, "containing
only RNA molecules that serve to catalyze the synthesis of themselves."4

The phrase "RNA World" stuck to the general hypothesis,
and has since come to denote the RNA-first, DNA-and-proteins-later
scenario depicted in Figure 1. The long-standing
"chicken-and egg" puzzle at the origin of life indeed
appeared amenable to a solution:

The primordial...conundrum -- which came first, informational
polynucleotides or functional polypeptides -- was obviated by
the simple but elegant compaction of both genetic information
and catalytic function into the same molecule.5

A second impetus to the RNA world hypothesis came from the
cluster of technical innovations now known generally as ribozyme
engineering. Naturally occuring RNA catalytic activities are
actually restricted to a small set of highly specialized reactions,
e.g., the processing of RNA transcripts primarily in eukaryotic
cells. However, ribozyme engineering, made possible by techniques
such as DNA sequencing, in vitro transcription and the
polymerase chain reaction [PCR]6,
allow molecular biologists to manipulate RNA to whatever extent
the molecule will allow. Thus, the catalytic repertoire of RNA
can be expanded beyond the naturally occurring activities -- in
the main, by two broad strategies of ribozyme engineering.

One strategy involves the direct modification of existing species
of ribozymes, to produce better or even novel catalysts. This
has been called the "rational design" approach. The
other strategy employs pools of short (often 50-100 nucleotide
units) randomized RNA molecules, which are subjected repeatedly
to a selection process designed to enhance the concentration of
RNA molecules with the desired functional activity. The few selected
molecules are then multiplied a million-fold or more by using
the polymerase chain reaction, which uses activated nucleotide
precursors and enzymes. This has been termed the "irrational
design" method.

Judging from the progress in ribozyme engineering in recent
years, it seems likely that new and improved types of RNA catalysts
will be produced in years ahead. Moreover, molecular biologists
may discover additional catalytic roles of RNA in living cells,
although the variety of such roles is not expected to rival that
of the protein enzymes. Thus, one might expect that the RNA World
hypothesis will continue to have supporters.

Yet beyond the immediate foreground of RNA World excitement
lies a disquieting landscape of chemical problems, largely ignored
in the recent literature on ribozyme engineering. As researchers
broaden their focus to include the chemical plausibility of the
RNA World itself, however7,
these difficulties cannot be avoided.

Furthermore, the relevance of ribozyme engineering to naturalistic
theories of the origin of life is doubtful at best, primarily
because of the necessity for intelligent intervention in the synthesis
of the randomized RNA; then again in the selection of a few functional
RNA molecules out of that mixture; then, finally, in the amplification
of those few functional RNA molecules [see
box, "What Do Ribozyme Engineering Experiments Really
Tell Us About the Origin of Life?"].

Hubert Yockey, borrowing a metaphor from Jonathan Swift, suggests
that current origin-of-life research, including the RNA World
hypothesis, floats improbably in mid-air like the roof of a house
built by an architect of the Grand Academy of Lagado. This savant
had contrived a method of building houses by beginning at the
roof and working downwards. "The architect pointed out that
among the advantages of this procedure," Yockey notes8, "was that once the
roof was in place [before the walls or foundation] the rest of
the construction could proceed quickly and without interruption
by weather." That "roof" -- consisting in this
instance of tiles which represent the catalytic activities of
RNA -- may look solid to those believers in the existence of a
prebiotic RNA World. But is the roof really solid? Is it supported
by walls and a foundation?

Once one peers over the edge of the roof to look beneath, we
shall argue, the implausibility of the theoretical structure as
a whole is inescapable. In what follows, we present the key postulates
or presuppositions on which the RNA World hypothesis must rest
(see Figure 2). Each represents an unsolved
chemical problem, in every case well-known to origin-of-life researchers.
Unfortunately, in many articles on the RNA World, these problems
are often collapsed into the "prebiotic soup" and "self-assembly"
phases of the scenario, and receive no discussion. We suggest
that new discoveries about the catalytic activities of RNA should
be seen for what they really are: not elucidating prebiotic
processes on the early Earth, but rather as extending our knowledge
of the molecular biology of the cell in important ways (see below).

The relevance of catalytic RNA to the problem of the naturalistic
origin of life is, however, a different matter entirely.

We take heart in noting that, despite the frequent neglect
in much of the popular literature of the chemical difficulties
of the RNA World scenario, many of the scientists involved with
that hypothesis are quite candid in their assessment of the problems
associated with it. These are represented for instance by the
numerous contributors to The RNA World7.
Since the RNA World hypotheses are so broad, we will attempt to
break them down into somewhat narrower postulates. In this way
one may see more clearly some of the presuppositions that are
involved.

Problematic Chemical Postulates of the
RNA World Scenario

Postulate 1: There was a prebiotic pool of beta-D-ribonucleotides.

Beta-D-ribonucleotides (see Figure 2)
are compounds made up of a purine (adenine or guanine) or a pyrimidine
(uracil or cytosine) linked to the 1'-position of ribose in the
beta-configuration.

There is, in addition, a phosphate group attached to the 5'-position
of the ribose. For the four different ribonucleotides in this
prebiotic scenario, there would be hundreds of other possible
isomers.

But each of these four ribonucleotides is built up of three
components: a purine or pyrimidine, a sugar (ribose), and phosphate.
It is highly unlikely that any of the necessary subunits would
have accumulated in any more than trace amounts on the primitive
Earth. Consider ribose. The proposed prebiotic pathway leading
to this sugar, the formose reaction, is especially problematic9. If various nitrogenous
substances thought to have been present in the primitive ocean
are included in the reaction mixture, the reaction would not proceed.
The nitrogenous substances react with formaldehyde, the intermediates
in the pathways to sugars, and with sugars themselves to form
non-biological materials10.
Furthermore, as Stanley Miller and his colleagues recently reported,
"ribose and other sugars have suprisingly short half-lives
for decomposition at neutral pH, making it very unlikely that
sugars were available as prebiotic reagents."11

Or consider adenine. Reaction pathways proposed for the prebiotic
synthesis of this building block start with HCN in alkaline (pH
9.2) solutions of NH4OH.12
These reactions give small yields of adenine (e.g., 0.04%) and
other nitrogenous bases provided the HCN concentration is greater
than 0.01 M. However, the reaction mixtures contain a great variety
of nitrogenous substances that would interfere with the formose
reaction. Therefore, the conditions proposed for the prebiotic
synthesis of purines and pyrimidines are clearly incompatible
with those proposed for the synthesis of ribose. Moreover, adenine
is susceptible to deamination and ring-opening reactions (with
half-lives of about 80 years and 200 years respectively at 37º
C and neutral pH), making its prebiotic accumulation highly improbable13. This makes it difficult
to see how any appreciable quantities of nucleosides and nucleotides
could have accumulated on the primitive Earth. If the key components
of nucleotides (the correct purines and pyrimidines, ribose, and
phosphate) were not present, the possibility of obtaining a pool
of the four beta-D-ribonucleotides with correct linkages would
be remote indeed.

If this postulate, the first and most crucial assumption, is
not valid, however, then the entire hypothesis of an RNA World
formed by natural processes becomes meaningless.

Postulate 2: Beta-D ribonucleotides spontaneously form polymers
linked together by 3', 5'-phosphodiester linkages (i.e., they
link to form molecules of RNA; see figure 2).

Joyce and Orgel discuss candidly the problems with this postulate14. They note that nucleotides
do not link unless there is some type of activation of the phosphate
group. The only effective activating groups for the nucleotide
phosphate group (imidazolides, etc.), however, are those that
are totally implausible in any prebiotic scenario. In living organisms
today, adenosine-5'-triphosphate (ATP) is used for activation
of nucleoside phosphate groups, but ATP would not be available
for prebiotic syntheses. Joyce and Orgel note the possible use
of minerals for polymerization reactions, but then express their
doubts about this possibility15:

Whenever a problem in prebiotic synthesis seems intractable,
it is possible to postulate the existence of a mineral that catalyzes
the reaction...such claims cannot easily be refuted.

In other words, if one postulates an unknown mineral catalyst
that is not readily testable, it is difficult to refute the hypothesis.

Joyce and Orgel then note that if there were activation of
the phosphate group, the primary polymer product would have 5',
5'-pyrophosphate linkages; secondarily 2', 5'-phosphodiester linkages
-- while the desired 3',5'-phosphodiester linkages would be much
less abundant. However, all RNA known today has only 3',5'-phosphodiester
linkages, and any other linkages would alter the three-dimensional
structure and possibilities for function as a template or a catalyst.

Even waiving these obstacles, and allowing for minute amounts
of oligoribonucleotides, these molecules would have been rendered
ineffective at various stages in their growth by adding incorrect
nucleotides, or by reacting with the myriads of other substances
likely to have been present. Moreover, the RNA molecules would
have been continuously degraded by spontaneous hydrolysis and
other destructive processes operating on the primitive Earth16.

In brief, any movement in the direction of an RNA World on
a realistically-modeled early Earth would have been continuously
suppressed by destructive cross-reactions.

Postulate 3: A polyribonucleotide (i.e. RNA molecule), once
formed, would have the catalytic activity to replicate itself,
and a population of such self-replicating molecules could arise.

The difficulty with this postulate is evident in the following
quotation from Joyce and Orgel:

...it is assumed...that a magic catalyst existed to convert
the activated nucleotides to a random ensemble of polynucleotide
sequences, a subset of which had the ability to replicate. It
seems to be implicit that such sequences replicate themselves
but, for whatever reason, do not replicate unrelated neighbors.17

They refer to this as a component of "The Molecular Biologists
Dream," and discuss the difficulties inherent in such a view.
In order for a stable population of self-replicating RNA
molecules to arise -- a prerequisite for further evolution --
the RNA molecules must be able to replicate themselves with high
fidelity, or the sequence specificity which makes self-replication
possible at all will be lost. While "it is difficult to state
with certainty the minimum possible size of an RNA replicase ribozyme,"
Joyce and Orgel note, it seems unlikely that a structure with
fewer than 40 nucleotides would be sufficient. Suppose, then,
that "there is some 50-mer [RNA molecule of 50 nucleotides
length]," Joyce and Orgel speculate, that "replicates
with 90% fidelity. ... Would such a molecule be expected to occur
within a population of random RNAs?"

Perhaps: but one such self-replicating molecule will not suffice.

"Unless the molecule can literally copy itself,"
Joyce and Orgel note, "that is, act simultaneously as both
template and catalyst, it must encounter another copy of itself
that it can use as a template." Copying any given RNA in
its vicinity will lead to an error catastrophe, as the population
of RNAs will decay into a collection of random sequences. But
to find another copy of itself, the self-replicating RNA would
need (Joyce and Orgel calculate) a library of RNA that "far
exceeds the mass of the earth."18

In the face of these difficulties, they advise, one must reject

the myth of a self-replicating RNA molecule that arose de
novo from a soup of random polynucleotides. Not only is such
a notion unrealistic in light of our current understanding of
prebiotic chemistry, but it should strain the credulity of even
an optimist's view of RNA's catalytic potential. If you doubt
this, ask yourself whether you believe that a replicase ribozyme
would arise in a solution containing nucleoside 5'-diphosphates
and polynucleotide phosphorylase!19

Postulate 4: Self-replicating RNA molecules wouild have all
of the catalytic activities necessary to sustain a ribo-organism.

...one is forced to conclude that the last ribo-organism had
a relatively complex metabolism that included oxidation and reduction
reactions, aldol and Claison condensations, transmethylations,
porphyrin biosynthesis, and an energy metabolism based on nucleoside
phosphates, all catalyzed by riboenzymes...It should be noted
that this reconstruction cannot be weakened without losing much
of the logical and explanatory force of the RNA World model.

Although Benner et al. speak of the last "ribo-organism,"
surely the first ribo-organism would have required nearly
all of the same metabolic capabilities in order to survive. It
is also apparent that the scenario of Benner et al. would surely
include enclosing the ribozymes within a membrane with the ability
to transport ions and organic molecules across that membrane.

Anyone who is familiar with biochemistry would recognize that
it would take hundreds of different ribozymes, each with a particular
catalytic activity, to carry out the metabolic processes described
above. It should also be apparent that most of these metabolic
capabilities would have to be functional within a short period
of time (certainly not hundreds of years), in the same microscopic
region, or the ribo-organism would never survive.

When one recognizes that catalytic activities of RNA are just
as dependent upon specific sequences of nucleotides in RNA21 as protein enzymes
are of amino acid sequences, then the probability of postulate
4 being valid is seen to be vanishingly small.

Benner et al. note that the diverse catalytic properties of
enzymes often require coenzymes or prosthetic groups. They mention
particularly the iron-porphyrin, heme, and pyridoxal, but have
no suggestion how these (and other co-enzymes) could have functioned
in the catalytic activities of early RNA molecules.

The other unproven assumption of postulate 4 is that RNA molecules
initially had all of these suggested catalytic activities, but
nearly all of these activities have been subsequently lost. RNA
molecules with catalytic activity that are known today predominantly
have nuclease or nucleotidyl transferase activity with some minimal
esterase actitivy22.
There is no solid evidence that RNA molecules ever had the broad
range of catalytic activities suggested by Benner et al., even
though a number of the authors of The RNA World speak of
present-day RNA molecules as being vestiges of that early RNA
World.

Conclusion

We have more to learn about RNA, both in vivo (as used
by organisms) and in vitro, in terms of its chemistry generally
and functional properties in particular. RNA is a remarkable molecule.

The RNA World hypothesis is another matter. We see no grounds
for considering it established, or even promising, except perhaps
on the objectionable philosophical grounds of philosophical naturalism
(and its operational offspring, methodological naturalism), according
to which the best naturalistic hypothesis is perforce the hypothesis
to be accepted. We consider that historical biology should be
open to all empirical possibilities, including design -- and see
the molecular biological system of organisms, of which RNA is
so stunning a part, as exemplars of design.

We find ourselves, however, distinctly in the minority of biologists.
If design exists at all, it is a matter of subjective intuition,
the majority of our colleagues would claim, asserting with science
writer George Johnson that "the point of science is...to
explain the world through natural law."23

We would put the point rather differently. The point of science
is to explain the world, through natural laws or
whatever other causes best account for the phenomena at hand.

Philosopher of science Stephen Meyer captures the point well:

The (historical) question that must be asked about biological
origins is not "Which materialistic scenario will prove
adequate?" but "How did life as we know it actually
arise on earth?" Since one of the logically appropriate
answers to this latter question is that "Life was designed
by an intelligent agent that existed before the advent of humans,"
I believe it is anti-intellectual to exclude the "design
hypothesis" without consideration of all the evidence, including
the most current evidence, that would support it.24

Detecting design is not a matter of subjective intuition.25 To see design as a
real causal possibility, however, one must break free of the constraints
of naturalism.

10. Recently it
has been shown that reaction mixtures containing dilute glycoaldehyde
phosphate and formaldehyde or glyceraldehyde-2-phophate will
generate reasonably high yields of ribose 2,4-diphosphate and
a few other sugar phosphates in less amounts. See D. Muller,
S. Pitsch, A. Kittaka, E. Wagner, C.E. Wintner, and A. Eschenmoser,
"Chemie von alpha-aminonitrilen. Aldomerisierung von glykoaldehydphosphat
zu racemischen hexose- 2,4,6-triphosphaten und (in gegenwart
von formaldehyd) racemischen pentose 2,4-diphophaten:
rac.allose-2,4,6-triphosphat und rac.-ribose-2,4,-diphosphat
sind die reaktionshauptproduckte. Helv. Chim. Acta 73
(1990): 1410-1468; Joyce and Orgel, ibid. However, if these reactions
are not also run in the presence of amines and other nitrogenous
compounds (i.e., in chemical mixtures of the complexity proposed
for the "prebiotic soup"), their relevancy to the origin
of life is problematical.

21. T.R. Cech,
"Mechanism and Structure of a Catalytic RNA Molecule,"
in 40 Years of the Double Helix, The Robert A. Welch Foundation
37th Conference on Chemical Research, 1993, pp. 91-110; see also
T.R. Cech, "Structure and Mechanism of the Large Catalytic
RNAs: Group I and Group II Introns and Ribonuclease P,"
in The RNA World, eds. R.F. Gesteland and J.F. Atkins
(Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press,
1993), pp. 239-269.

25. See William
A. Dembski, "The Design Inference: Eliminating Chance Through
Small Probabilities," unpublished Ph.D. dissertation, 1995,
Department of Philosophy, University of Illinois-Chicago Circle.